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<TITLE>RE: [RASMB] time to reach equilibrium & protein stability</TITLE>
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<P><FONT FACE="Arial">Dear RASMBers,</FONT><FONT FACE="Times New Roman"> </FONT>
<BR><FONT FACE="Arial">the protein stability issue is most important in the context of reaching equilibrium. Getting a protein stable in a non physiological environment for many hours or even days is not trivial in many cases, when (auto)proteolytic cleavage or aggregation processes continuously modify molecular mass.</FONT><FONT FACE="Times New Roman"> </FONT></P>
<P><FONT FACE="Arial">Fortunately, in this case nature helps AUC, as small proteins which need more time to reach equilibrium are often more stable than large proteins. However, when stability can be improved by addition of glycerol or decreasing temperature, viscosity increases as well and with it the time to reach equilibrium. Optimization is therefore required sometimes.</FONT><FONT FACE="Times New Roman"> </FONT></P>
<P><FONT FACE="Arial">Prediction of time to reach equilibrium based on theoretical assumptions is very important to give an initial guess. However, when control profiles show that it takes much longer to reach equilibrium or when equilibrium is never achieved, one should investigate protein stability. Light scattering methods (dynamic and static) turned out very helpful in my lab for doing this and to obtain a first insight into some parameters (average diffusion coefficients or molecular masses) of the system. Such measurements are even faster than Sed velocity experiments...</FONT><FONT FACE="Times New Roman"> </FONT></P>
<P><FONT FACE="Arial">Best regards from Basel,</FONT><FONT FACE="Times New Roman"> </FONT>
<BR><FONT FACE="Arial">Hans-Joachim Schönfeld.</FONT><FONT FACE="Times New Roman"> </FONT>
<BR><FONT SIZE=2 FACE="Tahoma">============================================</FONT><FONT FACE="Times New Roman"><BR>
</FONT><FONT SIZE=2 FACE="Tahoma">Dr. Hans-Joachim Schönfeld</FONT><FONT FACE="Times New Roman"><BR>
</FONT><FONT SIZE=2 FACE="Tahoma">F. Hoffmann-La Roche Inc.</FONT><FONT FACE="Times New Roman"><BR>
</FONT><FONT SIZE=2 FACE="Tahoma">PRBD-E, B93/5.44</FONT><FONT FACE="Times New Roman"><BR>
</FONT><FONT SIZE=2 FACE="Tahoma">CH-4070 Basel</FONT><FONT FACE="Times New Roman"><BR>
</FONT><FONT SIZE=2 FACE="Tahoma">Switzerland</FONT><FONT FACE="Times New Roman"> </FONT>
<BR><FONT SIZE=2 FACE="Tahoma">Tel. (+41) 61 688 28 95</FONT><FONT FACE="Times New Roman"><BR>
</FONT><FONT SIZE=2 FACE="Tahoma">Fax. (+41) 61 688 90 60</FONT><FONT FACE="Times New Roman"><BR>
</FONT><A HREF="mailto:hans-j.schoenfeld@roche.com"><U><FONT COLOR="#0000FF" SIZE=2 FACE="Tahoma">mailto:hans-j.schoenfeld@roche.com</FONT></U></A><FONT FACE="Times New Roman"> </FONT>
<BR><FONT SIZE=2 FACE="Times New Roman">-----Original Message-----<BR>
<B>From:</B> rasmb-admin@server1.bbri.org [</FONT><A HREF="mailto:rasmb-admin@server1.bbri.org"><U><FONT COLOR="#0000FF" SIZE=2 FACE="Times New Roman">mailto:rasmb-admin@server1.bbri.org</FONT></U></A><FONT SIZE=2 FACE="Times New Roman">]</FONT><B> <FONT SIZE=2 FACE="Times New Roman">On Behalf Of</FONT></B> <FONT SIZE=2 FACE="Times New Roman">John Correia<BR>
</FONT><B><FONT SIZE=2 FACE="Times New Roman">Sent:</FONT></B><FONT SIZE=2 FACE="Times New Roman"> Wednesday, September 29, 2004 5:31 PM<BR>
</FONT><B><FONT SIZE=2 FACE="Times New Roman">To:</FONT></B><FONT SIZE=2 FACE="Times New Roman"> strauss@fmp-berlin.de; rasmb@server1.bbri.org<BR>
</FONT><B><FONT SIZE=2 FACE="Times New Roman">Subject:</FONT></B><FONT SIZE=2 FACE="Times New Roman"> Re: [RASMB] time to reach equilibrium<BR>
</FONT>
<BR><FONT FACE="Arial">Cheers</FONT><FONT FACE="Times New Roman"> </FONT>
<BR><FONT FACE="Arial">We published a paper in 1977 that predicted the equilibrium distribution from earlier scans:</FONT><FONT SIZE=2 FACE="Arial"></FONT> <FONT SIZE=2 FACE="Times New Roman">J.J. CORREIA, G.H. Weiss and D.A. Yphantis (1977). "An Extrapolation Method for Reducing Equilibrium Times in Sedimentation Equilibrium Systems," Biophysical J. 20, 153</FONT><FONT SIZE=2 FACE="MS Mincho"></FONT><FONT SIZE=2 FACE="Times New Roman">168. The point being, as Holger just mentioned, many samples aggregate with time, or display insolubility, instability. Many of us have observed this with proteins like tubulin which like to associate in polymorphic forms, and with many His tagged proteins. In many of these cases there is a slow loss of material from a "stable" exponential distribution. Information can still be extracted, with caution. Solutions to this problem have included change the pH, ionic strength, etc (obvious), drop the temperature, add reducing agent (TCEP has worked best in limiting cases), remove the His tag, go to a short column format (which speeds up the time dramatically (16-fold for a 4-fold reduction in column height).</FONT> </P>
<P><FONT SIZE=2 FACE="Times New Roman">I not only agree with Bo, I insist that you do Sed velocity before you waste time doing sed equilibrium - I suspect most people do this - furthermore for many systems the quantitative analysis</FONT><B> <FONT SIZE=2 FACE="Times New Roman">must</FONT></B><FONT SIZE=2 FACE="Times New Roman"> also come from Sed velocity - Sedanal, Sedphat, etc make this very inviting. In our lab sed velocity rules, ie we have no alternative!</FONT></P>
<P><FONT SIZE=2 FACE="Times New Roman">-------------------------------------------------------------------<BR>
Dr. John J. "Jack" Correia<BR>
Department of Biochemistry<BR>
University of Mississippi Medical Center<BR>
2500 North State Street<BR>
Jackson, MS 39216<BR>
(601) 984-1522<BR>
fax (601) 984-1501<BR>
email address: </FONT><A HREF="mailto:jcorreia@biochem.umsmed.edu"><U><FONT COLOR="#0000FF" SIZE=2 FACE="Times New Roman">jcorreia@biochem.umsmed.edu</FONT></U></A><FONT SIZE=2 FACE="Times New Roman"><BR>
homepage location: </FONT><A HREF="http://biochemistry.umc.edu/correia.html"><U><FONT COLOR="#0000FF" SIZE=2 FACE="Times New Roman">http://biochemistry.umc.edu/correia.html</FONT></U></A><BR>
<FONT SIZE=2 FACE="Times New Roman">dept homepage location: </FONT><A HREF="http://biochemistry.umc.edu/"><U><FONT COLOR="#0000FF" SIZE=2 FACE="Times New Roman">http://biochemistry.umc.edu/</FONT></U></A><BR>
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